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Protein structure prediction has emerged as a core technology for understanding biomolecules and their interactions. Here, we combine homology‐based structure prediction with molecular phylogenetic analysis to study the evolution of electrostatic membrane binding among the vertebrate synaptotagmin‐like protein (Slp) family. Slp family proteins play key roles in the membrane trafficking of large dense‐core secretory vesicles. Our previous experimental and computational study found that the C2A domain of Slp‐4 (also called granuphilin) binds with high affinity to anionic phospholipids in the cytoplasmic leaflet of the plasma membrane through a large positively charged protein surface centered on a cluster of phosphoinositide‐binding lysine residues. Because this surface contributes greatly to Slp‐4 C2A domain membrane binding, we hypothesized that the net charge on the surface might be evolutionarily conserved. To test this hypothesis, the known C2A sequences of Slp‐4 among vertebrates were organized by class (from mammalia to pisces) using molecular phylogenetic analysis. Consensus sequences for each class were then identified and used to generate homology structures, from which Poisson–Boltzmann electrostatic potentials were calculated. For comparison, homology structures and electrostatic potentials were also calculated for the five human Slp protein family members. The results demonstrate that the charge on the membrane‐binding surface is highly conserved throughout the evolution of Slp‐4, and more highly conserved than many individual residues among the human Slp family paralogs. Such molecular phylogenetic‐driven computational analysis can help to describe the evolution of electrostatic interactions between proteins and membranes which are crucial for their function. [ABSTRACT FROM AUTHOR]

Sanye Tablet (SYT) is a patent prescription widely used in treating T2D and pre-diabetes, especially T2D comorbid with hypertriglyceridemia, for many years in China. However, the underlying mechanism that accounts for the anti-diabetic potential of SYT by regulating lipid-related intermediates remains to be elucidated. This study aimed to investigate the mechanism of SYT on lipid metabolism and insulin sensitivity in high-fat diet (HFD)-induced obese mice by means of combining lipidomics and proteomics. The obese mice models were developed via HFD feeding for 20 consecutive weeks. Mice in the treatment group were given metformin and SYT respectively, and the effects of SYT on body weight, blood glucose, insulin sensitivity, fat accumulation in the organs, and pathological changes in the liver were monitored. Lipid metabolism was examined by lipidomics. Further determination of signaling pathways was detected by proteomics. The biological contributions of the compounds detected in SYT’s chemical fingerprint were predicted by network pharmacology. SYT treatment reduced body weight, inhibited viscera and hepatic steatosis lipid accumulation, and prevented insulin resistance. Furthermore, it was found that circulatory inflammatory cytokines were reduced by SYT treatment. In addition, lipidomics analysis indicated that SYT targets lipid intermediates, including diacylglycerol (DAG) and Ceramide (Cer). Mechanistically, SYT positively affected these lipid intermediates by suppressing liver lipogenesis via downregulation of SREBP1/ACC and the JAK/STAT signaling pathway. Our results predicted that astragalin and rosmarinic acid might regulate the JAK-STAT pathway by targeting PIM2 and STAT1, respectively, while paeoniflorin and rosmarinic acid were likely to regulate inflammatory responses by targeting TNFα, IL-6, and IL-4 during T2D. Overall, our study provides supportive evidence for the mechanism of SYT’s therapeutic effect on dysregulated lipid metabolism in diabesity.

Sanye Tablet (SYT) is a patent prescription widely used in treating T2D and pre-diabetes, especially T2D comorbid with hypertriglyceridemia, for many years in China. However, the underlying mechanism that accounts for the anti-diabetic potential of SYT by regulating lipid-related intermediates remains to be elucidated. This study aimed to investigate the mechanism of SYT on lipid metabolism and insulin sensitivity in high-fat diet (HFD)-induced obese mice by means of combining lipidomics and proteomics. The obese mice models were developed via HFD feeding for 20 consecutive weeks. Mice in the treatment group were given metformin and SYT respectively, and the effects of SYT on body weight, blood glucose, insulin sensitivity, fat accumulation in the organs, and pathological changes in the liver were monitored. Lipid metabolism was examined by lipidomics. Further determination of signaling pathways was detected by proteomics. The biological contributions of the compounds detected in SYT's chemical fingerprint were predicted by network pharmacology. SYT treatment reduced body weight, inhibited viscera and hepatic steatosis lipid accumulation, and prevented insulin resistance. Furthermore, it was found that circulatory inflammatory cytokines were reduced by SYT treatment. In addition, lipidomics analysis indicated that SYT targets lipid intermediates, including diacylglycerol (DAG) and Ceramide (Cer). Mechanistically, SYT positively affected these lipid intermediates by suppressing liver lipogenesis via downregulation of SREBP1/ACC and the JAK/STAT signaling pathway. Our results predicted that astragalin and rosmarinic acid might regulate the JAK-STAT pathway by targeting PIM2 and STAT1, respectively, while paeoniflorin and rosmarinic acid were likely to regulate inflammatory responses by targeting TNFα, IL-6, and IL-4 during T2D. Overall, our study provides supportive evidence for the mechanism of SYT's therapeutic effect on dysregulated lipid metabolism in diabesity. [ABSTRACT FROM AUTHOR]

Background & Objective: Soft tissue sarcomas (STS) constitute an uncommon and heterogeneous group of tumors of mesenchymal origin and various cytogenetic abnormalities ranging from distinct genomic rearrangements, such as chromosomal translocations and amplifications, to more intricate rearrangements involving multiple chromosomes. Fluorescence in situ hybridization (FISH) can be used to identify these chromosomal translocations and amplifications, and sub classify STS precisely. The current study aimed at investigating the usefulness of FISH, as a diagnostic ancillary aid, to detect cytogenetic abnormalities such as MDM2 (murine double minute 2) amplification and CHOP(C/EBP homologous protein) rearrangement in liposarcoma, as well as SYT (synaptotagmin) rearrangement in synovial sarcoma. Methods: The FISH technique was used to analyze 17 specimens of liposarcoma for MDM2 amplification and CHOP rearrangement, and 10 specimens of synovial sarcoma for SYT rearrangement. The subtypes of liposarcoma and synovial sarcomas were reclassified according to the FISH results and compared with those of the respective histological findings. Results: According to the FISH results in 17 liposarcoma cases, well-differentiated liposarcoma(WDLPS), dedifferentiated liposarcoma (DDLPS), and myxoidliposarcoma (MLPS)subtypes were 41%, 53%, and 6%, respectively. In different subtypes of liposarcoma, a total of 30% mismatches were observed between pathologic and cytogenetic results. According to the histological findings from FISH analysis, SYT rearrangement was found only in three out of 10 (30%) synovial sarcomas. Conclusion: The detection of cytogenetic abnormalities in patients with liposarcoma and synovial sarcoma by FISH technique provides an important objective tool to confirm sarcoma diagnosis and sub classification of specific sarcoma subtypes in such patients.

Protein structure prediction has emerged as a core technology for understanding biomolecules and their interactions. Here, we combine homology-based structure prediction with molecular phylogenetic analysis to study the evolution of electrostatic membrane binding among the vertebrate synaptotagmin-like protein (Slp) family. Slp family proteins play key roles in the membrane trafficking of large dense-core secretory vesicles. Our previous experimental and computational study found that the C2A domain of Slp-4 (also called granuphilin) binds with high affinity to anionic phospholipids in the cytoplasmic leaflet of the plasma membrane through a large positively charged protein surface centered on a cluster of phosphoinositide-binding lysine residues. Because this surface contributes greatly to Slp-4 C2A domain membrane binding, we hypothesized that the net charge on the surface might be evolutionarily conserved. To test this hypothesis, the known C2A sequences of Slp-4 among vertebrates were organized by class (from mammalia to pisces) using molecular phylogenetic analysis. Consensus sequences for each class were then identified and used to generate homology structures, from which Poisson-Boltzmann electrostatic potentials were calculated. For comparison, homology structures and electrostatic potentials were also calculated for the five human Slp protein family members. The results demonstrate that the charge on the membrane-binding surface is highly conserved throughout the evolution of Slp-4, and more highly conserved than many individual residues among the human Slp family paralogs. Such molecular phylogenetic-driven computational analysis can help to describe the evolution of electrostatic interactions between proteins and membranes which are crucial for their function., (© 2023 The Protein Society.)

Objective: Synovial sarcomas (SS) are rare soft tissue tumours defined by the SYT‐SSX fusion gene. The tumours are composed of mesenchymal cells with varying degrees of epithelial differentiation. Cytomorphological descriptive studies are limited to small series and single cases. In this study we systematically examined the cytological features of SS diagnosed at our institution. Methods: SS diagnosed by fine‐needle aspiration (FNA) cytology at our institution between 2006 and 2018 were reviewed by a panel of senior cytopathologists. Clinical and cytopathological characteristics were categorised and described. Results: A total of 38 SS FNAs were identified from 35 patients. The cytomorphology was uniform, presenting as highly cellular smears of clusters and individual cells with mixed round, oval and spindle cells. We frequently observed pericapillary arrangement and occasionally pink background stroma was seen. Glandular formation or epithelial components were identified in the majority of cases which on histology were subtyped as biphasic SS. Pleomorphism and mitoses were rare. Immunocytochemical analysis was frequently positive for vimentin, epithelial membrane antigen, Bcl2 and, in recent cases, TLE1. Pan‐cytokeratins and CK7 could occasionally be positive in biphasic cases. The diagnostic SYT‐SSX fusion gene was detected in all FNA specimens using polymerase chain reaction or fluorescence in situ hybridisation. Conclusions: SS have distinct and uniform cytopathological features. Molecular genetic analysis for SYT‐SSX are invaluable for diagnosing SS with FNA and should be implemented in cytopathological laboratories that routinely perform soft tissue diagnostics. Synovial sarcomas are rare soft tissue tumors defined by the SYT‐SSX fusion gene. In this study, we demonstrate that synovial sarcomas have distinct and homogenous cytopathologic features. Molecular genetic analysis for SYT‐SSX are invaluable for diagnosing SS with FNA and should be implemented in cytopathological laboratories which routinely perform soft tissue diagnostics. [ABSTRACT FROM AUTHOR]

The coordination of multiple metabolic activities in plants relies on an interorganelle communication network established through membrane contact sites (MCS). The MCS are maintained in transient or durable configurations by tethering structures which keep the two membranes in close proximity, and create chemical microdomains that allow localized and targeted exchange of small molecules and possibly proteins. The past few years have witnessed a dramatic increase in our understanding of the structural and molecular organization of plant interorganelle MCS, and their crucial roles in plant specialized functions including stress responses, cell to cell communication, and lipid transport. In this review we summarize recent advances in understanding the molecular components, structural organization, and functions of different plant-specific MCS architectures. [ABSTRACT FROM AUTHOR]

Synaptotagmin is a synaptic vesicle membrane protein which changes conformation upon Ca 2+ binding and triggers the fast neuroexocytosis that takes place at synapses. We have synthesized a series of peptides corresponding to the sequence of the cytosolic juxtamembrane domain of synaptotagmin, which is highly conserved among different isoforms and animal species, with or without either a hexyl hydrophobic chain or the hexyl group plus a fluorescein moiety. We show that these peptides inhibit neurotransmitter release, that they localize on the presynaptic membrane of the motor axon terminal at the neuromuscular junction and that they bind monophosphoinositides in a Ca 2+ -independent manner. Based on these findings, we propose that the juxtamembrane cytosolic domain of synaptotagmin binds the cytosolic layer of the presynaptic membrane at rest. This binding brings synaptic vesicles and plasma membrane in a very close apposition, favouring the formation of hemifusion intermediates that enable rapid vesicle fusion. [ABSTRACT FROM AUTHOR]

Synovial sarcoma (SS) is a malignant mesenchymal neoplasm with variable epithelial differentiation, with a propensity to occur in young adults and which can arise at almost any site. It is generally viewed and treated as a high-grade sarcoma. As one of the first sarcomas to be defined by the presence of a specific chromosomal translocation leading to the production of the SS18-SSX fusion oncogene, it is perhaps the archetypal “translocation-associated sarcoma,” and its translocation remains unique to this tumor type. Synovial sarcoma has a variety of morphologic patterns, but its chief forms are the classic biphasic pattern, of glandular or solid epithelial structures with monomorphic spindle cells and the monophasic pattern, of fascicles of spindle cells with only immunohistochemical or ultrastructural evidence of epithelial differentiation. However, there is significant morphologic heterogeneity and overlap with a variety of other neoplasms, which can cause diagnostic challenge, particularly as the immunoprofile is varied, SS18-SSX is not detected in 100% of SSs, and they may occur at unusual sites. Correct diagnosis is clinically important, due to the relative chemosensitivity of SS in relation to other sarcomas, for prognostication and because of the potential for treatment with specific targeted therapies in the near future. We review SS, with emphasis on the diagnostic spectrum, recent immunohistochemical and genetic findings, and the differential diagnosis. [ABSTRACT FROM AUTHOR]

Abstract: The effects of palladium (Pd) on Sm0.2Ce0.8O2−δ coated Sr0.92Y0.08TiO3−δ (SDC/SYT) anodes were investigated for H2 and CH4 fuels. The electrochemical oxidations of both H2 and CH4 were accelerated by Pd impregnation. Moreover, Pd in the SDC/SYT (Pd-SDC/SYT) anode improved the cell performance by a factor of approximately 2 for H2 and 1.5 for CH4. The open circuit voltage (OCV) of the wet CH4 fuel increased with increasing temperature for both the SDC/SYT anode cell and Pd-SDC/SYT anode cell, which differs from that of the H2 fuel. Notably, the OCV values of the Pd-SDC/SYT anode cell using wet CH4 were much higher than those using wet H2. We observed differing potentials for the reformed gases after the out-of-cell catalyst experiment, and the CH4 fuel with the Pd-SDC catalyst layer agreed well with the OCVs of the Pd-SDC/SYT anode cell with directly introduced wet CH4 fuel. These results indicate the OCVs were higher than the theoretical values based on electrochemical hydrogen oxidation at increased temperatures in the Pd-SDC/SYT anode cell because of the lower water partial pressure caused by the increased steam reformation activity of Pd. [Copyright &y& Elsevier]

Abstract: Reduced insulin release has been linked to defect exocytosis in β-cells. However, whether expression of genes suggested to be involved in the exocytotic process (exocytotic genes) is altered in pancreatic islets from patients with type 2 diabetes (T2D), and correlate to insulin secretion, needs to be further investigated. Analysing expression levels of 23 exocytotic genes using microarray revealed reduced expression of five genes in human T2D islets (χ 2 =13.25; p <0.001). Gene expression of STX1A, SYT4, SYT7, SYT11, SYT13, SNAP25 and STXBP1 correlated negatively to in vivo measurements of HbA1c levels and positively to glucose stimulated insulin secretion (GSIS) in vitro in human islets. STX1A, SYT4 and SYT11 protein levels correspondingly decreased in human T2D islets. Moreover, silencing of SYT4 and SYT13 reduced GSIS in INS1-832/13 cells. Our data support that reduced expression of exocytotic genes contributes to impaired insulin secretion, and suggest decreased expression of these genes as part of T2D pathogenesis. [Copyright &y& Elsevier]

Abstract: Yttria-doped strontium titanium oxide (Sr0.92Y0.08TiO3−δ ; SYT) was investigated as an alternative anode material for solid oxide fuel cells (SOFCs). The SYT synthesized by the Pechini method exhibits excellent phase stability during the cell fabrication processes and SOFC operation and good electrical conductivity (about 0.85 S/cm, porosity 30%) in reducing atmosphere. The performance of SYT anode is characterized by slow electrochemical reactions except for the gas-phase diffusion reactions. The cell performance with the SYT anode running on methane fuel was improved about 5 times by SDC film coating, which increased the number of reaction sites and also accelerated electrochemical reaction kinetics of the anode. In addition, the SDC-coated SYT anode cell was stably operated for 900 h with methane. These results show that the SDC-coated SYT anode can be a promising anode material for high temperature SOFCs running directly on hydrocarbon fuels. [Copyright &y& Elsevier]

Synovial sarcomas arising in unexpected locations may lead to diagnostic challenges. In this report, we describe 3 cases of synovial sarcoma that manifested clinically as primary pericardial lesions. All 3 cases occurred in men in their fourth decade. Fever, cough, chest pain, and chest distress were the most common symptoms. Histologically, 2 of the tumors were spindle cell monophasic, and 1 tumor was biphasic. By immunohistochemical studies, the tumor cells were positive for cytokeratins and epithelial membrane antigen. In addition, the tumor cells displayed focal immunoreactivity for calretinin, cytokeratin 5/6, and HBME-1, resulting in the initial interpretations of malignant mesotheliomas. None of the 3 cases were diagnosed correctly until subsequent molecular cytogenetic assays demonstrated the presence of SYT gene rearrangements. As there are overlapping morphologic features between pericardial synovial sarcoma and mesothelioma, molecular analysis is essential for differential diagnoses. [ABSTRACT FROM AUTHOR]

USP6 rearrangement is the most common genetic abnormality in primary aneurysmal bone cyst, and SS18 rearrangement has not been previously described in any type of tumor where synovial sarcoma was excluded from the differential diagnosis. We report a case of solid aneurysmal bone cyst in which fluorescence in situ hybridization (FISH) analysis indicated rearrangements of both USP6 and SS18, but histologic features were consistent with aneurysmal bone cyst throughout the lesion. Reverse-transcription polymerase chain reaction (RT-PCR) for the SS18-SSX1 and SS18-SSX2 translocations, identity testing, and SS18 FISH were performed on cytogenetic monolayer cultures and formalin-fixed paraffin-embedded (FFPE) tissue. Genomic microarray, FISH, and immunohistochemistry were performed on follow-up studies of the FFPE specimen. The karyotype was 45,X,add(X)(p11.2),add(4)(q13),add(8)(p21),-13,add(17)(p11.2),add(18)(q11.2) in all 20 cells analyzed from monolayer cultures. The karyotype showed no cytogenetically visible alterations of chromosomal regions harboring known partners for USP6. Metaphase FISH with a commercial SS18 break-apart probe showed translocation of the 5′ portion of the SS18 probe to the short arm of the derivative X, as is observed in synovial sarcoma. RT-PCR showed no evidence of a SS18-SSX fusion, and immunohistochemistry was negative for TLE1, EMA, and cytokeratin AE1/3 expression. FISH on FFPE sections with a custom break-apart probe flanking USP6 showed evidence for a USP6 rearrangement throughout the tumor (25–50%). FISH on FFPE sections with a commercial SS18 break-apart FISH probe showed more variable results (0–50% split signals). There was no evidence of a SS18-USP6 fusion by FISH or RT-PCR. A molecular inversion probe array revealed a deletion encompassing the entire SS18 gene and its promoter, as well as portions of the region targeted by the commercial SS18 FISH probe. In conclusion, results obtained from commercially available FISH probes may occasionally yield misleading results. In this case, the SS18 rearrangement by FISH resulted from a complex rearrangement of 18q11.2 with a deletion of the SS18 gene. The translocation partner for USP6 remains unknown in this case. [Copyright &y& Elsevier]

Abstract: The SYT proto-oncoprotein (also known as SS18) is a gene expression regulator conserved across species. Although its biological function is still unknown, the importance of SYT as a housekeeping protein is illustrated by the lethal phenotype of SYT-null embryos. Notably, SYT is a component of the synovial sarcoma-associated translocation product, the SYT–SSX oncogene. SYT was previously reported as a mediator of cell adhesion. In the present study we show that SYT possesses distinct domains that control MDCK cyst formation in three-dimensional collagen cultures. While the carboxy-half of SYT, the QPGY domain, is required for cyst growth, the amino-terminal region appears to exert on this process a regulatory effect. Further analysis suggested that the purinergic G protein-coupled P2Y receptor signaling is involved in SYT-induced cystogenesis. Activation of this cascade is due to facilitation of ATP release in the extracellular space of polarized MDCK cells by SYT. These studies allow us to begin to understand the vital role of SYT in controlling epithelial morphogenesis and might explain the lethality of its loss in the developing embryo. [Copyright &y& Elsevier]

The development of the flat morphology of leaf blades is dependent on the control of cell proliferation as well as cell expansion. Each process has a polarity with respect to the longitudinal and transverse axes of the leaf blade. However, only a few regulatory components of these processes have been identified to date. We have characterized two genes from Arabidopsis thaliana: ANGUSTIFOLIA3 ( AN3), which encodes a homolog of the human transcription coactivator SYT, and GROWTH-REGULATING FACTOR5 ( AtGRF5), which encodes a putative transcription factor. AN3 is identical to GRF-INTERACTING FACTOR1 ( AtGIF1). The an3 and atgrf5 mutants exhibit narrow-leaf phenotypes due to decreases in cell number. Conversely, cell proliferation in leaf primordia is enhanced and leaves grow larger than normal when AN3 or AtGRF5 is overexpressed. Both genes are expressed in leaf primordia, and in the yeast two-hybrid assay, the gene products were found to interact with each other through their N-terminal domains. These results suggest that AN3 and AtGRF5 act together and are required for the development of appropriate leaf size and shape through the promotion and/or maintenance of cell proliferation activity in leaf primordia. [ABSTRACT FROM AUTHOR]

The human SYT–SSX gene has two splicing isoforms (type N and I), the latter of which contains an additional insertion of 93 bases. In the present study, we found increased transcriptional activity of the SYT–SSX type I protein in luciferase assay. When the SYT–SSX cDNAs were transfected to NIH3T3 cells, the type I transformant grew faster than the type N transformant. Furthermore, we evaluated the isoform ratio of the SYT or SYT–SSX transcripts in various tissues. Our results suggest that the SYT–SSX type I protein plays a critical role in the tumorigenesis of synovial sarcomas through increased transcriptional activity. [Copyright &y& Elsevier]

Abstract Background Although synovial sarcoma is the 3rd most commonly occurring mesenchymal tumor in young adults, usually with a highly aggressive clinical course; remarkable differences can be seen regarding the clinical outcome. According to comparative genomic hybridization (CGH) data published in the literature, the simple and complex karyotypes show a correlation between the prognosis and clinical outcome. In addition, the connection between DNA ploidy and clinical course is controversial. The aim of this study was using a fine-tuning interpretation of our DNA ploidy results and to compare these with metaphase high-resolution CGH (HR-CGH) results. Methods DNA ploidy was determined on Feulgen-stained smears in 56 synovial sarcoma cases by image cytometry; follow up was available in 46 cases (average: 78 months). In 9 cases HR-CGH analysis was also available. Results 10 cases were found DNA-aneuploid, 46 were DNA-diploid by image cytometry. With fine-tuning of the diploid cases according to the 5c exceeding events (single cell aneuploidy), 33 cases were so called "simple-diploid" (without 5c exceeding events) and 13 cases were "complex-diploid"; containing 5c exceeding events (any number). Aneuploid tumors contained large numbers of genetic alterations with the sum gain of at least 2 chromosomes (A-, B- or C-group) detected by HR-CGH. In the "simple-diploid" cases no or few genetic alterations could be detected, whereas the "complex-diploid" samples numerous aberrations (equal or more than 3) could be found. Conclusions Our results show a correlation between the DNA-ploidy, a fine-tuned DNA-ploidy and the HR-CGH results. Furthermore, we found significant correlation between the different ploidy groups and the clinical outcome (p < 0.05).

Background & Objective: Soft tissue sarcomas (STS) constitute an uncommon and heterogeneous group of tumors of mesenchymal origin and various cytogenetic abnormalities ranging from distinct genomic rearrangements, such as chromosomal translocations and amplifications, to more intricate rearrangements involving multiple chromosomes. Fluorescence in situ hybridization (FISH) can be used to identify these chromosomal translocations and amplifications, and sub classify STS precisely. The current study aimed at investigating the usefulness of FISH, as a diagnostic ancillary aid, to detect cytogenetic abnormalities such as MDM2 (murine double minute 2 ) amplification and CHOP (C/EBP homologous protein) rearrangement in liposarcoma, as well as SYT (synaptotagmin ) rearrangement in synovial sarcoma., Methods: The FISH technique was used to analyze 17 specimens of liposarcoma for MDM2 amplification and CHOP rearrangement, and 10 specimens of synovial sarcoma for SYT rearrangement. The subtypes of liposarcoma and synovial sarcomas were reclassified according to the FISH results and compared with those of the respective histological findings., Results: According to the FISH results in 17 liposarcoma cases, well-differentiated liposarcoma(WDLPS), dedifferentiated liposarcoma (DDLPS), and myxoidliposarcoma (MLPS)subtypes were 41%, 53%, and 6%, respectively. In different subtypes of liposarcoma, a total of 30% mismatches were observed between pathologic and cytogenetic results. According to the histological findings from FISH analysis, SYT rearrangement was found only in three out of 10 (30%) synovial sarcomas., Conclusion: The detection of cytogenetic abnormalities in patients with liposarcoma and synovial sarcoma by FISH technique provides an important objective tool to confirm sarcoma diagnosis and sub classification of specific sarcoma subtypes in such patients., Competing Interests: The authors declare that they have no conflict of interest.

The COMPLEXIN (CPX) proteins play a critical role in synaptic vesicle fusion and neurotransmitter release. Previous studies demonstrated that CPX functions in both activation of evoked neurotransmitter release and inhibition/clamping of spontaneous synaptic vesicle fusion. Here we report a new cpx mutant in Drosophila melanogaster, cpx(1257), revealing spatially defined and separable pools of CPX which make distinct contributions to the activation and clamping functions. In cpx(1257), lack of only the last C-terminal amino acid of CPX is predicted to disrupt prenylation and membrane targeting of CPX. Immunocytochemical analysis established localization of wild-type CPX to active zone (AZ) regions containing neurotransmitter release sites as well as broader presynaptic membrane compartments including synaptic vesicles. Parallel biochemical studies confirmed CPX membrane association and demonstrated robust binding interactions of CPX with all three SNAREs. This is in contrast to the cpx(1257) mutant, in which AZ localization of CPX persists but general membrane localization and, surprisingly, the bulk of CPX-SNARE protein interactions are abolished. Furthermore, electrophysiological analysis of neuromuscular synapses revealed interesting differences between cpx(1257) and a cpx null mutant. The cpx null exhibited a marked decrease in the EPSC amplitude, slowed EPSC rise and decay times and an increased mEPSC frequency with respect to wild-type. In contrast, cpx(1257) exhibited a wild-type EPSC with an increased mEPSC frequency and thus a selective failure to clamp spontaneous release. These results indicate that spatially distinct and separable interactions of CPX with presynaptic membranes and SNARE proteins mediate separable activation and clamping functions of CPX in neurotransmitter release., (Copyright © 2013 Elsevier Inc. All rights reserved.)